NOTE - see middle of page for previous weeks' schedules and things to catch up on.
Definitions: 'cubby' = VDS cabinet in back of lab where all of our reagents are; 'pigeon hole' = the shelf for your lab notebook over by the window Timeline F12
Wikispaces check on every other Sunday (Bi-weekly)
100113-
VDSers - please close laptops and place them back on the shelves when you are done using them.
- Thanks Dr. B
101513 - VDSers - we have 'orange' tape labelled some TE and Elution buffers by the Nanodrop. You want to keep those at the nanodrop and not use them to elute your sample from MidiPrep and MiniPrep and PCR CleanUp. Use the ones in the kits for elution because the ones near the nanodrop aren't that clean anymore.
10/28/13 - For the Ligand Prep Protocol in Virtual Screening, the Maestro program is not starting. So, use the ICM Ligand Preparation Steps from the LigPrep Protocols to prepare your control ligands. GDocs/Protocols/VirtualScreenignProtocols/Protocol_LigandPrep_VDS_v1_6.docx
Springers, for Virtual Screening only screen: your Control Ligands, CB306 (306 ligs), InHouseCompounds (~40 ligs), NIHClinicalCollection (446 ligs), HF9PlatesPlates5_9.sdf (400 ligs)
10/30/13 - T4 DNA Polymerase is now from Fermentas (instead of NEB). Use the Fermentas buffer with it.
110413 - Note about Maybridge Compounds (HitFinder 9 library)
Remove the final letters. e.g. KM10410SC needs to be changed to ---> KM10410 Also Ryan Scientific has info on Maybridge ligands
11/13/13
There is a revised ProtocolProteinExpression_VDS_Fall13_v5.doc on GDocs/ProteinProtocols
Surrogate Enzyme: -- use YopH (the staff made a new plate in the fridge)
11/25/13
Inhibitors to use for Phosphatases (e.g. the surrogate enzymes)
Chembridge ID:
5250098
5153298
5852635
- don't use the 50 mM in DMSO
- USE the 5 mM in DMSO/Water (the mentors will have made some of these for you)
They are in the 'Drugs' drawer in the back of the lab near our Cubby (near the yellow fire cabinet)
This Week
This Week
SPRINGERSWEEK 15
Finish to protein expression, purification, characterization protocols
Finish virtual screening
Enzyme Assay protocol
Inhibition Assay protocol
See the Mentor schedule for when we will have mentors on duty:
Finish up your Peer Reviews of
M&M Virtual
M&M Expression, Purification, Characterization
2-Dec Monday 3-Dec Tuesday 4-Dec Wednesday - last class
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section 5-Dec Thursday
Lab Cleanup Day - each person do 1 hr
Peer Reviews DUE on canvas
- cloning
- expression, purification, characterization
- virtual
6-Dec Friday
Lab Cleanup Day - each person do 1 hr
7-Dec Saturday
Final Lab Report Due @ 9pm
8-Dec Sunday
VDS Social - Dr. B's house 5pm RSVP Form
Wikispaces posting due - 9 pm
9-Dec Monday
Lab Notebook check - Monday night
Next Week
Next Week
SPRINGERS
VETERANS
ITEMS FOR LATER WEEKS:
LICpNIC-Bsa4cloningExerciseVDS_Fall13.docx
Set up target protein for Virtual Screening
- Make Homology Models
ICM Protocol
Dilute tail primers - make stock in TE and working dilution in water
-- store stock in VDS Primers 2013 Box
-- store working dilution in YOUR own box
Midi-prep your pNIC-Bsa4
After successful Midiprep, Submit to DNA Sequencing (pNIC-Bsa4- option B)
submit pNIC-Bsa4 to sequencing (pLICfor & pLICrev)
-- ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall13.doc
RE Digest w/BsaI of your pNIC-Bsa4, check on agarose gel
FIRST PRIORITY
TDRTargets Exercise
- enter info on spreadsheet
Choose target options
- submit your preferences to Target Selection Form Fall13
Once assigned a target
- enter info onto the TargetSelections Fall13 sheet on GDocs
- Oligo Primer Design
SECOND PRIORITY
Nanodrop plasmid DNA - ProtocolNanoDropVDS.doc
-- Plasmid to work with: pGBR22
Submit to DNA Sequencing (pGBR22 - option A)
THIRD PRIORITY (can do next week too)
Analyze DNA Sequence
-- (only proceed to this step after TDRTargets Exercise and Target Selections Form)
Create a GDocs folder in /StudentFolders - FirstnameLastInitial (e.g. WilliamB)
Visit your Wikispaces research page under Research Pages 2013
Create your own freezer box, share it half & half with someone else
upload final primers to Google Docs/VDS/Misc/Primers/PrimerPlateOrderFall2013
post final primers to Wikispaces page
Add content to YOUR research page Research Pages 2013
Put a line and 'Week 1' Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
29-Aug Thursday
4:30 - 6:30 pm
Help Session: PyMol Refresher
in WEL 2.114
cloning & expression work
30-Aug Friday
cloning & expression work
4:00 - 6:00 pm
Help Session: PyMol Refresher
in WEL 2.114
Add content to YOUR research page Research Pages 2013
Put a line and 'Week 1' Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
31-Aug Saturday 1-Sep Sunday
PyMol Refresher - due next Wed
PRIORITY:
Oligo Primer Design
ProtocolPCR_PrimerDesign_OverlapAssemblyVDS_Fall13.doc
Analyze DNA Sequence
ProtocolPrimerDilutionVDS_Fall13.docx
- Dilute PCR primers - practice cloning
-- M13for, M13Rev, SP6, T7 promoter, pLIC-for, pLIC-rev
-- make working dilution in water from 100uM stock.
-- Store working dilution in YOUR own box.
PCR 'VDS PCR' - practice cloning
- ProtocolPCRVDS.xls
RE Digest - practice cloning
Tail Primer Design - order your own Tail Primers (as a Team)
WEEK 2 2-Sep Monday Labor Day - We have mentors on duty from 10-7 pm
Oligo Primer Design
Nanodrop & Submit to DNA Sequencing
PCR
Tail Primer Design (4 - 6 pm) in Wet Lab on laptops
6-Sep Friday
Tail Primer Design (3 - 6 pm) in Wet Lab on laptops
Write in your hours into the notebook - (also add in last week's) NOTE: hours will be totaled and graded bi-weekly
Add content to YOUR research page Research Pages 2013
Put a line and 'Week 2' Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
Lab Notebook check - Friday night
What Goes Where:
Oligo Design - upload output file to GDocs, paste Oligo sequence into your Target page, in Notebook too
Nanorop - 2 images for Wiki & protocol/images in Notebook
Submit to DNA - protocol in Notebook, if have result - put in Notebook too
Analyze DNA Sequence - upload results to GDocs (put the protocol in your Notebook - results can stay in GDocs)
PCR & run on an agarose gel - images to Wiki & protocol/images in Notebook
7-Sep Saturday 8-Sep Sunday
Target page updates due - 9 pm
Wikispaces posting due - 9 pm
ITEMS FOR THE WEEK:
Primary, Secondary, PCR Squared
Setting up your Target for Virtual Screening.
WEEK 2 2-Sep Monday Labor Day - We have mentors on duty from 10-7 pm
3-Sep Tuesday Target Meeting open times 1:30 - 3:00 pm
- swing by Dr. B's office (see list above for those that sill need to meet)
4-Sep Wednesday
2 - 3pm - Lab Group Meeting -
- NOTE: changed to GEA 125 a.k.a. 'world's coolest classroom'
Write in your hours into the notebook - (also add in last week's) NOTE: hours will be totaled and graded bi-weekly
Add content to YOUR research page Research Pages 2013
Put a line and 'Week 2' Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
Lab Notebook check - Friday night
7-Sep Saturday 8-Sep Sunday
Wikispaces posting due - 9 pm
WEEK 3
PCR 'VDS PCR' - Re Do if it failed (see Dr. B if you're not sure)
RE Digest -
Tail Primer Design - order your own Tail Primers (as a Team)
- we will have appointed times for this( see Thurs & Fri Google Calendar) ProtocolPCR_PrimerDesignTails_for_pNIC-Bsa4CloningFall13.doc
Tail Primer ordering through IDT website (some instructions show at top of this page) Teams that still need to order their Tail Primers (should be done Mon or Tues) https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodDJlZDlneXMtb1JuREV0MUZQYXU4YkE#gid=0
Jessica, Qasim, Kelly
Aditi, Grant T.
Gautam W., Anita V., Ariel C.
Keely, Nicole
Brandy, D'Ondria, Caroline C.
Jesus, James, Jiaqi
After you have ordered tail primers and have made your Oligo Mix, you can start the first step of cloning: Primary PCR - (~1.5hrs to prepare and run PCR, then save in -20degC or run agarose gel check)
16-Sep Monday 17-Sep Tuesday 18-Sep Wednesday
2 - 3pm - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
4-5 PM: PAI 2.14 site-specific training (OH 102) (just show up in Lab then)
19-Sep Thursday
Primary PCR
Make LB
Make LB Agar plates (Kan only)
20-Sep Friday
Primary PCR
Make LB
Make LB Agar plates (Kan only)
21-Sep Saturday 22-Sep Sunday
Wikispaces posting due - 9 pm
Over the next 2 weeks: Primary PCR on your target
Make 160ml of LB - can do in Teams, but each person makes 160ml
Make 6x (LB+Kan) plates - need 6 for the whole Team
ProtocolLBAgarPlatesVDS.doc (not the Kan/Suc ones)
WEEK 4
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Primary & Secondary PCR's - should work
PCR squared - if Primary & Secondary PCR worked
PyMol Refresher - can count as 2 hours of lab time
Make 160ml of LB - can do in Teams, but each person makes 160ml
Make 6x (LB+Kan) plates - need 6 for the whole Team
ProtocolLBAgarPlatesVDS.doc (not the Kan/Suc ones)
23-Sep Monday 3-4 PM: PAI 2.14 site-specific training (OH 102)
24-Sep Tuesday 25-Sep Wednesday 1-2 PM: PAI 2.14 site-specific training (OH 102)
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
NOTE: we have Saturday lab times this week 11:30 - 5:00 pm
29-Sep Sunday
NEXT STEPS if other things completed:
Transformation of pNIC-Bsa4 (as a Team - should end up with 2 plates/person)
start growing up pNIC-Bsa4 overnight in 160ml of LB + Kan
spin down next morning (save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you dont' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours
Midi-prep (to make lots of plasmid that we will need for cloning)
ProtocolTransformationforPlasmidPrep_pNIC_Bsa4_VDS_v3.doc
Do this protocol in Teams.
NOTE: which antibiotic will you need
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol)
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
*
WEEK 5
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Transformation of pNIC-Bsa4 (as a Team - should end up with 2 plates/person)
- ProtocolTransformationforPlasmidPrep_pNIC_Bsa4_VDS_v3.doc
Do this protocol in Teams.
NOTE: which antibiotic will you need
Grow up pNIC-Bsa4 overnight in 160ml of LB + Kan
--spin down next morning (save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you don't' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours til you can spin it in the afternoon. Pellets can be frozen and stored til next day or so.
Midi-prep (to make lots of plasmid that we will need for cloning)
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol), gel check
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
Grow up pNIC-Bsa4 overnight in 160ml of LB + Kan
--spin down next morning
(save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you don't' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours til you can spin it in the afternoon. Pellets can be frozen and stored til next day or so.
Midi-prep (to make lots of plasmid that we will need for cloning)
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol), gel check
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
You don't have to do all of these protocols right away - but you should aim to have your Homology model created this week and have started on Control Ligands selection (the first part of the Virtual Screening Protocol).
-- see the sign up spreadsheet above for times
Target meeting with your mentor - email or contact you mentor on Piazza to set up a time for meeting.
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol Day 2
-- ProtocolProteinExpression_VDS_Fall13_v4.doc
30-Oct Wednesday 1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc' Journal Club Class Slides posted to Piazza resources section
Cloning Materials & Methods due at 3 am - (i.e Wed night)
-- see Piazza resources to post questions and get help
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v4.doc
7-Nov Thursday 8-Nov Friday 9-Nov Saturday
No lab notebook check
10-NovSunday
Peer Review of M&M Cloning is due @ 11:59 PM
- you will have 2 to review
- make 5 constructive criticisms
- make 2 compliments
WEEK 12
We will have more widely spread mentor coverage to help you with protein expression, etc.
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH andFtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v5.doc
-Finish Control Ligands Docking
- start on 1st Libraries after you have finished the Control Ligands docking
move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH andFtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v5.doc
- Finish these Libraries after you have finished the Control Ligands docking
Peer Reviews for both of these have been assigned:
M&M Virtual
M&M Expression
WEEK 13
Move to surrogate enzyme if you have not already done so.
The Chembridge Inhibitors have come in for the 1st round of ordering (FabG, 6PGDH, NDM-1)
- mentors will make Stocks of each to 50 mM
- each person doing an inhibition assays should do Working Stock solubility test
- then make a Working stock for inhibition assays.
-- for directions, see GDocs/Misc/Compounds/instructionsdilutingcompounds
For Enzyme Assays, see general Enzyme Assay protocol on GDocs. Enzyme Assay First Attempts
- Melissa, Jason, Alyssa, ? Aliquoting Substrates for Assays:
Jason - ?
- we will do a split class on the Monday before Thanksgiving. see the Schedule below for Week 14
EARLY CLASS: 2-3 pm Monday - GEA 100 (unfortunately not the 'WCC' - but still very cool)
SERENA Z
ANITA V
ANTONIO G
JACQUELINE E
SEO K
KEVIN E
MELISSA H
OSCAR V
Renee
KATHERINE V
WILLIAM E
JESSICA N
JULIA C
PRIYA P
BRANDY C
Mohammad A
Shelby F-G
MANUEL Z
IMRAN Z
NICOLE W
KEELY W
CAROLINE C
D'ONDRIA P
Jiaqi Z
LATE CLASS: 3-4 pm Monday - GEA 100 (unfortunately not the 'WCC' - but still very cool)
BRENDAN C
MARIANNA U
ARIEL C
GRACE T
ASHLEE W
HYUN-YOUNG L
RAMIRO R
VICTORIA G
Madeline J
KAVYA K
FENG G
GAUTAM W
DANIEL D
JENSEN G
JAMES T
NICOLET F
Jesus De La O
GRANT T
ADITI S
JASON P
Alyssa K
Target meetings: 1st one completed: Tony & Renee, Serena & Madeline & Brendan, Oscar & Priya, Alyssa & Stephanie, Kavya & Vicky, Nicolet, Katherine V. , Melissa & Jason, Ashlee & Marianna, Young & Grace , Ariel & Anita
Primer Ordering Instructions (Sept. 13th 2013)
Everyone designs their primer - but we will only order one person's set.
Be sure you use the correct DNA Works output file to get your codon optimized DNA sequence (not just the CDS).
-- If you are use a Summer A,B,D,D, or E plate - you need to get your DNA Works Output file from Misc/Primers/DNAWorks_OutputSummer13
-- If you are use a Fall A or B plate - you need to get your DNA Works Output file from Misc/Primers/DNAWorks_OutputFall13
Save the Word doc of your primer design into your GDocs
Print Word doc and paste into your Lab Notebook
Crosscheck your sequence with your Teammates
Put the sequences of the Tail Primers on your team's target page.
-- Enter your name for the person whose primers we will order - once you have designed the primers
Put the info and sequence on the Target Spreadsheet https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodDJlZDlneXMtb1JuREV0MUZQYXU4YkE#gid=0
Order your primers
-- go to the ICMB core site (see our Targets Spreadsheet for the link)
-- find the IDT link
-- enter our username: VDS (all caps) and password: same as the password to get on to the laptops buildingname+labroomnumber
--Products/DNA/RNASynthesis/CustomDNAOligos
--select 2 for the Multiple Entry
-- leave defaults for Scale and Purification
-- enter your 2 primers and the Prefixes.
-- save to Wishlist, Dr. B will order when we have several of them.
TIMELINE FOR RESEARCH
Schedule of things we will do over the semesterNOTE - see middle of page for previous weeks' schedules and things to catch up on.
Definitions: 'cubby' = VDS cabinet in back of lab where all of our reagents are; 'pigeon hole' = the shelf for your lab notebook over by the window
Timeline F12
Wikispaces check on every other Sunday (Bi-weekly)
Weeks layout
Week 13 & 14 18-Nov - 1-Dec
Week 11 & 12 4-Nov - 17-Nov
Week 9 & 10 21-Oct - 3-Nov
Week 5 & 6 23-Sep - 6-Oct
Week 3 & 4 9-Sep - 22-Sep
Week 1 & 2 26-Aug - 8-Sep
- (can put in Safety section of VDS sign-in binder in lab)
Split Class Information
100113-
VDSers - please close laptops and place them back on the shelves when you are done using them.
- Thanks Dr. B
101513 - VDSers - we have 'orange' tape labelled some TE and Elution buffers by the Nanodrop. You want to keep those at the nanodrop and not use them to elute your sample from MidiPrep and MiniPrep and PCR CleanUp. Use the ones in the kits for elution because the ones near the nanodrop aren't that clean anymore.
101813 - Research Progress Table
ResearchProgressF13
10/28/13 - For the Ligand Prep Protocol in Virtual Screening, the Maestro program is not starting. So, use the ICM Ligand Preparation Steps from the LigPrep Protocols to prepare your control ligands. GDocs/Protocols/VirtualScreenignProtocols/Protocol_LigandPrep_VDS_v1_6.docx
Springers, for Virtual Screening only screen: your Control Ligands, CB306 (306 ligs), InHouseCompounds (~40 ligs), NIHClinicalCollection (446 ligs), HF9PlatesPlates5_9.sdf (400 ligs)
10/30/13 - T4 DNA Polymerase is now from Fermentas (instead of NEB). Use the Fermentas buffer with it.
110413 - Note about Maybridge Compounds (HitFinder 9 library)
- Maybridge – Login as VDSclass@gmail.com Password is painter214
Remove the final letters. e.g. KM10410SC needs to be changed to ---> KM10410Also Ryan Scientific has info on Maybridge ligands
11/13/13
Surrogate Enzyme:
-- use YopH (the staff made a new plate in the fridge)
11/25/13
Inhibitors to use for Phosphatases (e.g. the surrogate enzymes)
Chembridge ID:
- USE the 5 mM in DMSO/Water (the mentors will have made some of these for you)
They are in the 'Drugs' drawer in the back of the lab near our Cubby (near the yellow fire cabinet)
This Week
This Week
Finish to protein expression, purification, characterization protocols
Finish virtual screening
Enzyme Assay protocol
Inhibition Assay protocol
See the Mentor schedule for when we will have mentors on duty:
Finish up your Peer Reviews of
M&M Virtual
M&M Expression, Purification, Characterization
Guidelines for Final Research Report:
posted to GDocs/Results&Data/ResearchReport_Materials&Methods_Guidelines
VDSFall13_GuideFinalReportonTarget_VDS_v2.doc
2-Dec Monday
Lab Notebook check - Monday night (Springers only)
3-Dec Tuesday
4-Dec Wednesday - last class
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
5-Dec Thursday
Lab Cleanup Day - each person do 1 hr
Peer Reviews DUE on canvas
- cloning
- expression, purification, characterization
- virtual
6-Dec Friday
Lab Cleanup Day - each person do 1 hr
7-Dec Saturday
Final Lab Report Due @ 9pm
8-Dec Sunday
VDS Social - Dr. B's house 5pm
RSVP Form
Wikispaces posting due - 9 pm
9-Dec Monday
Lab Notebook check - Monday night
Finish up your Peer Reviews of
M&M Virtual
M&M Expression, Purification, Characterization
Enzyme Assays, Inhibition Assays
Guidelines for Final Research Report:
posted to GDocs/Results&Data/ResearchReport_Materials&Methods_Guidelines
VDSFall13_GuideFinalReportonTarget_VDS_v2.doc
2-Dec Monday
3-Dec Tuesday
4-Dec Wednesday - last class
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
5-Dec Thursday
Lab Cleanup Day - each person do 1 hr
Peer Reviews DUE on canvas
- cloning
- expression, purification, characterization
- virtual
6-Dec Friday
Lab Cleanup Day - each person do 1 hr
7-Dec Saturday
Final Lab Report Due @ 9pm
8-Dec Sunday
VDS Social - Dr. B's house 5pm
RSVP Form
Wikispaces posting due - 9 pm
9-Dec Monday
Lab Notebook check - Monday night
Next Week
Next Week
LICpNIC-Bsa4cloningExerciseVDS_Fall13.docx
Set up target protein for Virtual Screening
- Make Homology Models
ICM Protocol
Dilute tail primers - make stock in TE and working dilution in water
-- store stock in VDS Primers 2013 Box
-- store working dilution in YOUR own box
Midi-prep your pNIC-Bsa4
After successful Midiprep, Submit to DNA Sequencing (pNIC-Bsa4- option B)
submit pNIC-Bsa4 to sequencing (pLICfor & pLICrev)
-- ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall13.doc
RE Digest w/BsaI of your pNIC-Bsa4, check on agarose gel
Lab Safety training
http://vdsstream.wikispaces.com/Required+Lab+Safety
FIRST PRIORITY
TDRTargets Exercise
- enter info on spreadsheet
Choose target options
- submit your preferences to Target Selection Form Fall13
Once assigned a target
- enter info onto the TargetSelections Fall13 sheet on GDocs
- Oligo Primer Design
SECOND PRIORITY
Nanodrop plasmid DNA - ProtocolNanoDropVDS.doc
-- Plasmid to work with: pGBR22
Submit to DNA Sequencing (pGBR22 - option A)
THIRD PRIORITY (can do next week too)
Analyze DNA Sequence
-- (only proceed to this step after TDRTargets Exercise and Target Selections Form)
Create a GDocs folder in /StudentFolders - FirstnameLastInitial (e.g. WilliamB)
Visit your Wikispaces research page under Research Pages 2013
Create your own freezer box, share it half & half with someone else
SignUps FirstWeeks Fall13 - interactive sheet
as a static sheet (so you can see it better):
WEEK 1
28-Aug Wednesday
2 - 3pm - Lab Group Meeting - BUR 108
3:30 --> ~ 5:30 pm
TDR Targets Exercise & Target Selection Form
in WEL 2.114
29-Aug Thursday
2:30 - 4:30 pm
TDR Targets Exercise & Target Selection Form
in WEL 2.114
4:30 - 6:30 pm (Wet Lab)
Nanodrop pGBR22, Submit to DNA Sequencing
30-Aug Friday
2 --> ~ 5pm
Overlap Primer Design
in WEL 2.114
Add content to YOUR research page
Research Pages 2013
Put a line and 'Week 1'
Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
31-Aug Saturday
1-Sep Sunday
continue cloning your targets
WEEK 1
28-Aug Wednesday
2 - 3pm - Lab Group Meeting - BUR 108
cloning & expression work
29-Aug Thursday
4:30 - 6:30 pm
Help Session: PyMol Refresher
in WEL 2.114
cloning & expression work
30-Aug Friday
cloning & expression work
4:00 - 6:00 pm
Help Session: PyMol Refresher
in WEL 2.114
Add content to YOUR research page
Research Pages 2013
Put a line and 'Week 1'
Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
31-Aug Saturday
1-Sep Sunday
PyMol Refresher - due next Wed
Lab Safety training
http://vdsstream.wikispaces.com/Required+Lab+Safety
See the /FirstProcotols Folder in Google Docs
PRIORITY:
Oligo Primer Design
ProtocolPCR_PrimerDesign_OverlapAssemblyVDS_Fall13.doc
Analyze DNA Sequence
ProtocolPrimerDilutionVDS_Fall13.docx
- Dilute PCR primers - practice cloning
-- M13for, M13Rev, SP6, T7 promoter, pLIC-for, pLIC-rev
-- make working dilution in water from 100uM stock.
-- Store working dilution in YOUR own box.
PCR 'VDS PCR' - practice cloning
- ProtocolPCRVDS.xls
RE Digest - practice cloning
Tail Primer Design - order your own Tail Primers (as a Team)
WEEK 2
2-Sep Monday
Labor Day
- We have mentors on duty from 10-7 pm
Oligo Primer Design
Nanodrop & Submit to DNA Sequencing
PCR
3-Sep Tuesday
4-Sep Wednesday
2 - 3pm - Lab Group Meeting - NOTE: changed to GEA 125
a.k.a. 'world's coolest classroom'
Class Slides posted to Piazza resources section
5-Sep Thursday
Tail Primer Design (4 - 6 pm) in Wet Lab on laptops
6-Sep Friday
Tail Primer Design (3 - 6 pm) in Wet Lab on laptops
Write in your hours into the notebook - (also add in last week's)
NOTE: hours will be totaled and graded bi-weekly
Add content to YOUR research page
Research Pages 2013
Put a line and 'Week 2'
Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
Lab Notebook check - Friday night
What Goes Where:
Oligo Design - upload output file to GDocs, paste Oligo sequence into your Target page, in Notebook too
Nanorop - 2 images for Wiki & protocol/images in Notebook
Submit to DNA - protocol in Notebook, if have result - put in Notebook too
Analyze DNA Sequence - upload results to GDocs (put the protocol in your Notebook - results can stay in GDocs)
PCR & run on an agarose gel - images to Wiki & protocol/images in Notebook
7-Sep Saturday
8-Sep Sunday
Target page updates due - 9 pm
Wikispaces posting due - 9 pm
Primary, Secondary, PCR Squared
Setting up your Target for Virtual Screening.
WEEK 2
2-Sep Monday
Labor Day
- We have mentors on duty from 10-7 pm
3-Sep Tuesday
Target Meeting open times 1:30 - 3:00 pm
- swing by Dr. B's office (see list above for those that sill need to meet)
4-Sep Wednesday
2 - 3pm - Lab Group Meeting -
- NOTE: changed to GEA 125 a.k.a. 'world's coolest classroom'
Class Slides posted to Piazza resources section
PyMol Refresher - due
5-Sep Thursday
6-Sep Friday
Write in your hours into the notebook - (also add in last week's)
NOTE: hours will be totaled and graded bi-weekly
Add content to YOUR research page
Research Pages 2013
Put a line and 'Week 2'
Below that add your 'result' image for the week with brief commentary
This page will run in reverse chronological order - i.e. Newest stuff on top
Lab Notebook check - Friday night
7-Sep Saturday
8-Sep Sunday
Wikispaces posting due - 9 pm
PCR 'VDS PCR' - Re Do if it failed (see Dr. B if you're not sure)
RE Digest -
Tail Primer Design - order your own Tail Primers (as a Team)
- we will have appointed times for this( see Thurs & Fri Google Calendar)
ProtocolPCR_PrimerDesignTails_for_pNIC-Bsa4CloningFall13.doc
Open Lab times
See Google Calendar for Open Lab Times
9-Sep Monday
add full DNA works output file to Notebook
10-Sep Tuesday
11-Sep Wednesday
2 - 3pm - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
12-Sep Thursday
Tail Primer Design in wet lab - Google Calendar
Optional: get a head start by growing up pNIC-Bsa4 overnight in 160ml of LB + Kan
spin down on Friday
13-Sep Friday
Tail Primer Design in wet lab - Google Calendar
see safety training links on next week's section
14-Sep Saturday
15-Sep Sunday
PCRs, Cloning, Expression work
start aiming to have cloning done by late September.
Set up your Virtual Screening on your target and run control dockings
See Google Calendar for Open Lab Times
9-Sep Monday
10-Sep Tuesday
11-Sep Wednesday
2 - 3pm - Lab Group Meeting - GEA 125
Class Slides posted to Piazza resources section
12-Sep Thursday
13-Sep Friday
see safety training links on next week's section
14-Sep Saturday
15-Sep Sunday
See Google Calendar for wet lab times
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Take CW512 online
http://vdsstream.wikispaces.com/Required+Lab+Safety
Tail Primer ordering through IDT website (some instructions show at top of this page)
Teams that still need to order their Tail Primers (should be done Mon or Tues)
https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodDJlZDlneXMtb1JuREV0MUZQYXU4YkE#gid=0
Jessica, Qasim, Kelly
Aditi, Grant T.
Gautam W., Anita V., Ariel C.
Keely, Nicole
Brandy, D'Ondria, Caroline C.
Jesus, James, Jiaqi
After you have ordered tail primers and have made your Oligo Mix, you can start the first step of cloning: Primary PCR
- (~1.5hrs to prepare and run PCR, then save in -20degC or run agarose gel check)
16-Sep Monday
17-Sep Tuesday
18-Sep Wednesday
2 - 3pm - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
4-5 PM: PAI 2.14 site-specific training (OH 102) (just show up in Lab then)
19-Sep Thursday
Primary PCR
Make LB
Make LB Agar plates (Kan only)
20-Sep Friday
Primary PCR
Make LB
Make LB Agar plates (Kan only)
21-Sep Saturday
22-Sep Sunday
Wikispaces posting due - 9 pm
Over the next 2 weeks:
Primary PCR on your target
Make 160ml of LB - can do in Teams, but each person makes 160ml
Make 6x (LB+Kan) plates - need 6 for the whole Team
ProtocolLBAgarPlatesVDS.doc (not the Kan/Suc ones)
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Take CW512 online
http://vdsstream.wikispaces.com/Required+Lab+Safety
PCRs, Cloning, Expression work
start aiming to have cloning done by late September.
16-Sep Monday
17-Sep Tuesday
Target meeting times available with Dr. B
18-Sep Wednesday
2 - 3pm - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
4-5 PM: PAI 2.14 site-specific training (OH 102)
19-Sep Thursday
Target meeting times available with Dr. B
20-Sep Friday
21-Sep Saturday
22-Sep Sunday
Wikispaces posting due - 9 pm
See Google Calendar for wet lab times
See Targets Grading Spreadsheet page for missing items on Target page
Due This Week:
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Take CW512 online
http://vdsstream.wikispaces.com/Required+Lab+Safety
Primary & Secondary PCR's - should work
PCR squared - if Primary & Secondary PCR worked
PyMol Refresher - can count as 2 hours of lab time
Make 160ml of LB - can do in Teams, but each person makes 160ml
Make 6x (LB+Kan) plates - need 6 for the whole Team
ProtocolLBAgarPlatesVDS.doc (not the Kan/Suc ones)
23-Sep Monday
3-4 PM: PAI 2.14 site-specific training (OH 102)
24-Sep Tuesday
25-Sep Wednesday
1-2 PM: PAI 2.14 site-specific training (OH 102)
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
Journal Club article posted for next week
26-Sep Thursday
27-Sep Friday
28-Sep Saturday
NOTE: we have Saturday lab times this week 11:30 - 5:00 pm
29-Sep Sunday
NEXT STEPS if other things completed:
Transformation of pNIC-Bsa4 (as a Team - should end up with 2 plates/person)
start growing up pNIC-Bsa4 overnight in 160ml of LB + Kan
spin down next morning (save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you dont' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours
Midi-prep (to make lots of plasmid that we will need for cloning)
ProtocolTransformationforPlasmidPrep_pNIC_Bsa4_VDS_v3.doc
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol)
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
*
Show Up for Site Specific Training (OH 102) for Fall Semester (PAI 2.14 lab) - see times available on the Lab Safety Page
Take CW512 online
http://vdsstream.wikispaces.com/Required+Lab+Safety
Cloning should be completed by end or September
Virtual Work
23-Sep Monday
3-4 PM: PAI 2.14 site-specific training (OH 102)
24-Sep Tuesday
Virtual Set up Times with Dr. B
See Google Calendar for wet lab times
New Protein Expression Protocol posted to GDocs (v. 4)
New Protein Purification Protocol posted to GDocs
25-Sep Wednesday
1-2 PM: PAI 2.14 site-specific training (OH 102)
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
26-Sep Thursday
Virtual Set up Times with Dr. B
See Google Calendar for wet lab times
27-Sep Friday
28-Sep Saturday
29-Sep Sunday
See Google Calendar for wet lab times
PCR squared
Transformation of pNIC-Bsa4 (as a Team - should end up with 2 plates/person)
- ProtocolTransformationforPlasmidPrep_pNIC_Bsa4_VDS_v3.doc
Grow up pNIC-Bsa4 overnight in 160ml of LB + Kan
--spin down next morning (save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you don't' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours til you can spin it in the afternoon. Pellets can be frozen and stored til next day or so.
Midi-prep (to make lots of plasmid that we will need for cloning)
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol), gel check
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
30-Sep Monday
1-Oct Tuesday
Cool talk tonight at 6:30 - see Announcement page for details
http://bit.ly/14nfYau
2-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
Research Report Guidelines posted:
GDocs/Results&Data/ResearchReport/
GuidelinesResearchReportonTarget_VDS_Fall13.doc
3-Oct Thursday
4-Oct Friday
5-Oct Saturday - should have Sat hours (no game)
6-Oct Sunday
Wikispaces posting due - 9 pm
NEXT STEPS
proceed to cloning (if you have PCR product and your vector is prepared)
Cloning should be completed by end or September
- note: update Expression & Purification protocols were posted last week
Virtual Work
30-Sep Monday
1-Oct Tuesday
3-6pm Virtual Screening help with Dr. B and mentors (PAI 2.14)
Cool talk tonight at 6:30 - see Announcement page for details
http://bit.ly/14nfYau
2-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
4-7 pm Virtual Screening help with Dr. B and mentors (PAI 2.14)
https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodGNnQy1fTWZybTFMZ2YyV29xNXlRMkE&usp=sharing
Research Report Guidelines posted:
GDocs/Results&Data/ResearchReport/
GuidelinesResearchReportonTarget_VDS_Fall13.doc
3-Oct Thursday
1-3 pm Virtual Screening help with Dr. B
5-8 pm Virtual Screening help with Daniel, Urvashi, Max, Brandon (PAI 2.14)
4-Oct Friday
1-3 pm Virtual Screening help with Dr. B and mentors (PAI 2.14)
5-Oct Saturday - should have Sat hours (no game)
ACL
6-Oct Sunday
Wikispaces posting due - 9 pm
See Google Calendar for wet lab times
Grow up pNIC-Bsa4 overnight in 160ml of LB + Kan
--spin down next morning
(save in -20degC or proceed to Midi-Prep - 2-3hrs)
-- if you don't' have time to spin down in morning (~30-45 min), you can temporarily store your overnight growth flask in 4degC for a few hours til you can spin it in the afternoon. Pellets can be frozen and stored til next day or so.
Midi-prep (to make lots of plasmid that we will need for cloning)
Cut pNIC-Bsa4 with BsaI (part of pNIC-Bsa4 Cloning protocol), gel check
If you have PCR Squared product and cut pNIC-Bsa4 accepting vector, proceed to Cloning
7-Oct Monday
8-Oct Tuesday
9-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
10-Oct Thursday
11-Oct Friday
12-Oct Saturday
- should have Sat hours (no game)
No lab notebook for Springers this week
13-Oct Sunday
Mid Semester Research Report due (a.k.a. Report on your Target)
Cloning should be complete
Grow up positive clone overnight in DH5alpha in 160 ml of LB
Midi-prep (verify by DNA Sequencing, then store in 2 separate places, give a sample of it to Dr. B for Archival storage)
Transform positive clone into BL21(DE3) cells for expression
Virtual
- Control Docking should be complete
- Virtual Screening of 1st libraries should be started
See Spreadsheet above or Google Calendar for virtual help timesDue on Saturday (for Veterans) - LIC cloning exercise
LICpNIC-Bsa4cloningExerciseVDS_Fall13.docx
7-Oct Monday
8-Oct Tuesday
9-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
10-Oct Thursday
11-Oct Friday
12-Oct Saturday
- should have Sat hours (no game)
Lab Notebook check - Saturday night (Veterans Only)
DUE: LICpNIC-Bsa4cloningExerciseVDS_Fall13.docx
13-Oct Sunday
Mid Semester Research Report due (a.k.a. Report on your Target)
See Google Calendar for wet lab times
Continue cloning your target, once clones proceed to protein expression
Virtual
- Begin Virtual Screening setup (see meeting times with Dr. B)
- create homology model if needed for your target
- ProtocolHomologyModelVDS_v4.doc
You don't have to do all of these protocols right away - but you should aim to have your Homology model created this week and have started on Control Ligands selection (the first part of the Virtual Screening Protocol).Protocol_LigandPrep_VDS_v1_6.docx
ProtocolVirtualScreenYOURTarget_VDS_Fall13.doc
-- see the sign up spreadsheet above for times
Target meeting with your mentor - email or contact you mentor on Piazza to set up a time for meeting.
14-Oct Monday
15-Oct Tuesday
16-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
17-Oct Thursday
18-Oct Friday
19-Oct Saturday
Lab Notebook check - Saturday night (Springers only)
DUE: LICpNIC-Bsa4cloningExerciseVDS_Fall13.docx
20-Oct Sunday
Wikispaces posting due - 9 pm
PyMol Image Contest
expression trials
Virtual
14-Oct Monday
15-Oct Tuesday
16-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
17-Oct Thursday
18-Oct Friday
19-Oct Saturday
No lab notebook for Veterans this week
20-Oct Sunday
Wikispaces posting due - 9 pm
PyMol Image Contest
See Google Calendar for wet lab times
Finish cloning, transform into BL21 cells
Meet with your Mentor for your Target Team
Start your virtual screening control docking
21-Oct Monday
22-Oct Tuesday
23-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
24-Oct Thursday
25-Oct Friday
PyMol Image Voting Form:
http://vdsstream.wikispaces.com/PyMolImageContest_F13
26-Oct Saturday
No lab notebook check
27-Oct Sunday
Homology Model due and Control Ligands
Cloning Materials & Methods due POSTPONED UNTIL Wed 10/30 at 3 am
Expression and enzyme assays test
Continue virtual screening on CB Diversity library
21-Oct Monday
22-Oct Tuesday
23-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
24-Oct Thursday
25-Oct Friday
PyMol Image Voting Form:
http://vdsstream.wikispaces.com/PyMolImageContest_F13
26-Oct Saturday
No lab notebook check
27-Oct Sunday
Control Ligands Docking due
Start CB306 and Chembridge Diversity
Cloning Materials & Methods due POSTPONED UNTIL Wed 10/30 at 3 am
use this link to edit and enter your name:
https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodGNnQy1fTWZybTFMZ2YyV29xNXlRMkE&usp=sharing
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol Day 2
-- ProtocolProteinExpression_VDS_Fall13_v4.doc
Control Ligands Docking
28-Oct Monday
29-Oct Tuesday
PyMol Image Voting Form:
http://vdsstream.wikispaces.com/PyMolImageContest_F13
30-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Journal Club
Class Slides posted to Piazza resources section
Cloning Materials & Methods due at 3 am - i.e. Wed night
-- see Piazza resources to post questions and get help
31-Oct Thursday
1-Nov Friday
Control Ligands Docking Due
2-Nov Saturday
No lab notebook check
3-Nov Sunday
Wikispaces posting due - 9 pm
Protein PurificationEnzyme Assay Trials
Virtual ScreeningCB Diversity (50K ligs) screening Due: - will order compounds at end of week-- Oscar, Priya, Serena, Madeline, Brendan, Vicky, Kavya,
CB306 Library Screening Due:- Grace, Melissa, Jason,
Aliquoting Substrates for Assays:
Oscar - yes
Vicky - yes
Kavya (?)
Melissa - yes
Priya
Serena - yes
Brendan - yes
Madeline - yes
Grace -
Jason -
WEEK 10
28-Oct Monday
29-Oct TuesdayPyMol Image Voting Form:
http://vdsstream.wikispaces.com/PyMolImageContest_F13
30-Oct Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Journal Club
Class Slides posted to Piazza resources section
Cloning Materials & Methods due at 3 am - (i.e Wed night)
-- see Piazza resources to post questions and get help
31-Oct Thursday
1-Nov Friday
2-Nov Saturday
Lab Notebook check - Saturday night (Veterans Only)
3-Nov Sunday
Wikispaces posting due - 9 pm
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v4.doc
-Finish Control Ligands Docking
- start on 1st Libraries
---InHouseCompounds (~40 ligs)
---CB306 (306 ligs)
4-Nov Monday
5-Nov Tuesday
Journal Club Q's from last meeting are due tomorrow in Class
6-Nov Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
7-Nov Thursday
8-Nov Friday
9-Nov Saturday
Lab Notebook check - Saturday night (Springers only)
10-NovSunday
Peer Review of M&M Cloning is due @ 11:59 PM
- you will have 2 to review
- make 5 constructive criticisms
- make 2 compliments
Enzyme Assay First Attempts- Grace, Melissa, Jason, Alyssa,
Aliquoting Substrates for Assays:
Jason -
Virtual Screening - ALL---InHouseCompounds (~40 ligs)
---CB306 (306 ligs)
---NIHClinicalCollection (446 ligs),
---HF9PlatesPlates5_9.sdf (400 ligs)
4-Nov Monday
5-Nov Tuesday
Journal Club Q's from last meeting are due tomorrow in Class
6-Nov Wednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
7-Nov Thursday
8-Nov Friday
9-Nov Saturday
No lab notebook check
10-NovSunday
Peer Review of M&M Cloning is due @ 11:59 PM
- you will have 2 to review
- make 5 constructive criticisms
- make 2 compliments
We will have more widely spread mentor coverage to help you with protein expression, etc.
After your 3rd cloning attempt, move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v5.doc
-Finish Control Ligands Docking
- start on 1st Libraries after you have finished the Control Ligands docking
---InHouseCompounds (~40 ligs) -use 1 processor
---CB306 (306 ligs) - use 1 processor
---NIHClinicalCollection (446 ligs), use 1 processor
---HF9PlatesPlates5_9.sdf (400 ligs) - use 1 processor
Journal Club paper posted for next week:
http://vdsstream.wikispaces.com/Journal+Club+Fall13
11-NovMonday
12-NovTuesday
13-NovWednesday
Dr. B in during the morning - if you need to ask questions
NO CLASS - DR. B to ABRCMS
Journal Paper for next week is posted to
vdsstream.wikispaces.com/Journal+Club+Fall13
14-NovThursday
15-NovFriday
16-NovSaturday
No lab notebook check
17-NovSunday
Virtual M&M due
-- see Piazza resources to post questions and get help
Guidelines:
https://drive.google.com/file/d/0B_Gl3lMyhDsoSDFsNjJvVENXcGs/edit?usp=sharing
Wikispaces posting due - 9 pm
For Enzyme Assays, see general Enzyme Assay protocol on GDocs.
Enzyme Assay First Attempts
- Melissa, Jason, Alyssa,
Aliquoting Substrates for Assays:
Jason -
Virtual Screening - ALL
-- finish libraries
---InHouseCompounds (~40 ligs)
---CB306 (306 ligs)
---NIHClinicalCollection (446 ligs),
---HF9PlatesPlates5_9.sdf (400 ligs)
Journal Club paper posted for next week:
http://vdsstream.wikispaces.com/Journal+Club+Fall13
11-NovMonday
12-NovTuesday
13-NovWednesday
Dr. B in during the morning - if you need to ask questions
NO CLASS - DR. B to ABRCMS
Journal Paper for next week is posted to
vdsstream.wikispaces.com/Journal+Club+Fall13
14-NovThursday
15-NovFriday
16-NovSaturday
No lab notebook check
17-NovSunday
Virtual M&M due
-- see Piazza resources to post questions and get help
Guidelines:
https://drive.google.com/file/d/0B_Gl3lMyhDsoSDFsNjJvVENXcGs/edit?usp=sharing
Wikispaces posting due - 9 pm
move to surrogate enzyme target
Print protocol and talk to Mentor or Dr. B about scheduling
Plates for YopH and FtHap are in the 4degC fridge
Proceed to protein expression protocol (Start on Day 2)
-- ProtocolProteinExpression_VDS_Fall13_v5.doc
- Finish these Libraries after you have finished the Control Ligands docking
---InHouseCompounds (~40 ligs) -use 1 processor
---CB306 (306 ligs) - use 1 processor
---NIHClinicalCollection (446 ligs), use 1 processor
---HF9PlatesPlates5_9.sdf (400 ligs) - use 1 processor
Journal Club paper posted:
http://vdsstream.wikispaces.com/Journal+Club+Fall13
18-NovMonday
19-NovTuesday
20-NovWednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
21-NovThursday
22-NovFriday
23-NovSaturday
No lab notebook check
24-NovSunday
Expression, Purification, Characterization M&M due
-- see Piazza resources to post questions and get help
Guidelines:
https://drive.google.com/file/d/0B_Gl3lMyhDsoWldaOEN5eHZNZDg/edit?usp=sharing
Peer Reviews for both of these have been assigned:
M&M Virtual
M&M Expression
Move to surrogate enzyme if you have not already done so.
The Chembridge Inhibitors have come in for the 1st round of ordering (FabG, 6PGDH, NDM-1)
- mentors will make Stocks of each to 50 mM
- each person doing an inhibition assays should do Working Stock solubility test
- then make a Working stock for inhibition assays.
-- for directions, see GDocs/Misc/Compounds/instructionsdilutingcompounds
For Enzyme Assays, see general Enzyme Assay protocol on GDocs.
Enzyme Assay First Attempts
- Melissa, Jason, Alyssa, ?
Aliquoting Substrates for Assays:
Jason - ?
Virtual Screening - ALL
-- finish libraries
---InHouseCompounds (~40 ligs)
---CB306 (306 ligs)
---NIHClinicalCollection (446 ligs),
---HF9PlatesPlates5_9.sdf (400 ligs)
Journal Club paper posted:
http://vdsstream.wikispaces.com/Journal+Club+Fall13
18-NovMonday
19-NovTuesday
20-NovWednesday
1:30 pm (early) 2:30 pm (late) - Lab Group Meeting - GEA 125 - a.k.a. the 'wcc'
Class Slides posted to Piazza resources section
21-NovThursday
22-NovFriday
23-NovSaturday
Lab Notebook check - Saturday night (Veterans Only)
24-NovSunday
Expression M&M due
-- see Piazza resources to post questions and get help
Guidelines:
https://drive.google.com/file/d/0B_Gl3lMyhDsoWldaOEN5eHZNZDg/edit?usp=sharing
Peer Reviews for both of these have been assigned:
M&M Virtual
M&M Expression
Finish to protein expression, purification, characterization protocols
Finish virtual screening
Enzyme Assay protocol
Inhibition Assay protocol
Finish up your Peer Reviews of
M&M Virtual
M&M Expression, Purification, Characterization
25-NovMonday
Lab Group Meeting - GEA 100 (Turkey Week shuffle)
2:00 pm (early)
3:00 pm (late) -
Class Slides posted to Piazza resources section
26-NovTuesday
27-NovWednesday
28-NovThursday
-- mandatory time out of lab - go eat a turkey
29-NovFriday
30-NovSaturday
n/a
1-Dec Sunday
Finish up your Peer Reviews of
M&M Virtual
M&M Expression, Purification, Characterization
Enzyme Assays, Inhibition Assays
25-NovMonday
Lab Group Meeting - GEA 100 (Turkey Week shuffle)
2:00 pm (early)
3:00 pm (late) -
Class Slides posted to Piazza resources section
26-NovTuesday
27-NovWednesday
28-NovThursday
-- mandatory time out of lab - go eat a turkey
29-NovFriday
30-NovSaturday
No lab notebook check
1-Dec Sunday
Turkey Week Shuffle
- we will do a split class on the Monday before Thanksgiving. see the Schedule below for Week 14EARLY CLASS: 2-3 pm Monday - GEA 100 (unfortunately not the 'WCC' - but still very cool)
KEELY W
D'ONDRIA P
LATE CLASS: 3-4 pm Monday - GEA 100 (unfortunately not the 'WCC' - but still very cool)
Target meetings:
1st one completed: Tony & Renee, Serena & Madeline & Brendan, Oscar & Priya, Alyssa & Stephanie, Kavya & Vicky, Nicolet, Katherine V. , Melissa & Jason, Ashlee & Marianna, Young & Grace , Ariel & Anita
Primer Ordering Instructions (Sept. 13th 2013)
Everyone designs their primer - but we will only order one person's set.
Be sure you use the correct DNA Works output file to get your codon optimized DNA sequence (not just the CDS).
-- If you are use a Summer A,B,D,D, or E plate - you need to get your DNA Works Output file from Misc/Primers/DNAWorks_OutputSummer13
-- If you are use a Fall A or B plate - you need to get your DNA Works Output file from Misc/Primers/DNAWorks_OutputFall13
Save the Word doc of your primer design into your GDocs
Print Word doc and paste into your Lab Notebook
Crosscheck your sequence with your Teammates
Put the sequences of the Tail Primers on your team's target page.
-- Enter your name for the person whose primers we will order - once you have designed the primers
Put the info and sequence on the Target Spreadsheet
https://docs.google.com/spreadsheet/ccc?key=0AvGl3lMyhDsodDJlZDlneXMtb1JuREV0MUZQYXU4YkE#gid=0
Order your primers
-- go to the ICMB core site (see our Targets Spreadsheet for the link)
-- find the IDT link
-- enter our username: VDS (all caps) and password: same as the password to get on to the laptops buildingname+labroomnumber
--Products/DNA/RNASynthesis/CustomDNAOligos
--select 2 for the Multiple Entry
-- leave defaults for Scale and Purification
-- enter your 2 primers and the Prefixes.
-- save to Wishlist, Dr. B will order when we have several of them.